US20130295574A1 - Method for Isolation of Nucleic Acid Containing Particles and Extraction of Nucleic Acids Therefrom - Google Patents

Method for Isolation of Nucleic Acid Containing Particles and Extraction of Nucleic Acids Therefrom Download PDF

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US20130295574A1
US20130295574A1 US13/883,673 US201113883673A US2013295574A1 US 20130295574 A1 US20130295574 A1 US 20130295574A1 US 201113883673 A US201113883673 A US 201113883673A US 2013295574 A1 US2013295574 A1 US 2013295574A1
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nucleic acids
nucleic acid
particles
rna
extracted
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Johan K. Skog
Leileata M. Russo
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Exosome Diagnostics Inc
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Exosome Diagnostics Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D21/00—Separation of suspended solid particles from liquids by sedimentation
    • B01D21/26—Separation of sediment aided by centrifugal force or centripetal force
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D21/00—Separation of suspended solid particles from liquids by sedimentation
    • B01D21/26—Separation of sediment aided by centrifugal force or centripetal force
    • B01D21/262—Separation of sediment aided by centrifugal force or centripetal force by using a centrifuge

Definitions

  • the present invention relates to the general fields of nucleic acid extraction from a biological sample, particularly the isolation of nucleic acid-containing particles from body fluids and extraction of nucleic acids from the isolated particles.
  • Exosomes Small microvesicles shed by cells are often described as “exosomes” (Thery et al., 2002). Exosomes are reported as having a diameter of approximately 30-100 nm and are shed from many different cell types under both normal and pathological conditions (Thery et al., 2002). Exosomes are classically formed from the inward invagination and pinching off of the late endosomal membrane. This results in the formation of a multivesicular body (MVB) laden with small lipid bilayer vesicles, each of which contains a sample of the parent cell's cytoplasm (Stoorvogel et al., 2002). Fusion of the MVB with the cell membrane results in the release of these exosomes from the cell, and their delivery into the blood, urine, cerebrospinal fluid, or other bodily fluids.
  • MVB multivesicular body
  • cell-derived microvesicles are formed by directly budding off of the cell's plasma membrane, are usually larger in size than exosomes, and like exosomes, also contain a sample of the parent cell's cytoplasm (Cocucci et al., 2009) (Orozco and Lewis, 2010).
  • WO 2009/100029 describes, among other things, the use of nucleic acids extracted from microvesicles in GBM patient serum for medical diagnosis, prognosis and therapy evaluation.
  • WO 2009/100029 also describes the use of nucleic acids extracted from microvesicles in human urine for the same purposes.
  • the use of nucleic acids extracted from microvesicles is considered to potentially circumvent the need for biopsies, highlighting the enormous diagnostic potential of microvesicle biology (Skog et al., 2008).
  • a method of magnetic activated cell sorting is described in a paper by Taylor and Gercel-Taylor (Taylor and Gercel-Taylor, 2008).
  • a method of nanomembrane ultrafiltration concentration is described in a paper by Cheruvanky et al. (Cheruvanky et al., 2007).
  • a method of Percoll gradient isolation is described in a publication by Miranda et. al (Miranda et al., 2010). Further, microvesicles may be identified and isolated from bodily fluid of a subject by a microfluidic device (Chen et al., 2010).
  • An object of the present invention is therefore to provide a method for quick and easy isolation of nucleic acid-containing particles from biological samples such as body fluids and extraction of high quality nucleic acids from the isolated particles.
  • the method of the invention may be suitable for adaptation and incorporation into a compact device or instrument for use in a laboratory or clinical setting, or in the field.
  • the present invention is based on our discovery that low speed centrifugation can be used to pellet particles from a biological sample and extract high quality nucleic acids from the particles.
  • the invention is a method for extracting nucleic acids by isolating nucleic acid-containing particles from a biological sample by one or more centrifugation procedures at a speed not exceeding about 200,000 g, performing one or more steps to mitigate adverse factors that prevent or might prevent high quality nucleic acid extraction; and extracting nucleic acids from the isolated particles.
  • the centrifugation procedures are performed at speeds of about 2,000 g to about 200,000 g. In other embodiments, the centrifugation procedures are performed at speeds not exceeding about 50,000 g. In still other embodiments, the centrifugation procedures are performed at speeds not exceeding about 20,000 g.
  • the method is used to extract nucleic acids from microvesicles, RNA-protein complexes, DNA-protein complexes, or a combination of any of microvesicles, RNA-protein complexes, and DNA-protein complexes.
  • the biological sample is a body fluid, for example, a serum or a urine sample from a subject.
  • the subject for example, can be a human or other mammal.
  • the extracted nucleic acids can be RNA, DNA, or both RNA and DNA.
  • the nucleic acids thus extracted contain one or more polynucleotides which are more than 90% homologous to a nucleic acid sequence corresponding to EGFR, BRAF, KLK3, 18S, GAPDH, HPRT1, GUSB, ACTB, B2M, RPLP0, HMBS, TBP, PGK1, UBC, PPIA, ALCAM, C5AR1, CD160, CD163, CD19, CD1A, CD1C, CD1D, CD2, CD209, CD22, CD24, CD244, CD247, CD28, CD37, CD38, CD3D, CD3G, CD4, CD40, CD40LG, CD5, CD6, CD63, CD69, CD7, CD70, CD72, CD74, CD79A, CD79B, CD80, CD83, CD86, CD8A, CD8B, CD96, CHST10, COL1A1, COL1A2, CR2, CSF1R, CTLA4, DPP4, ENG, FAS,
  • 18S rRNA and 28S rRNA are detectable in the extracted nucleic acids.
  • the ratio of the amount of 18S rRNA to the amount of 28S rRNA as detected in the extracted nucleic acids is about 0.5 to about 1.0. In other instances, the ratio of the amount of 18S rRNA to the amount of 28S rRNA as detected in the extracted nucleic acids is about 0.5.
  • the step of performing one or more steps to mitigate adverse factors is achieved by treating the biological sample and/or the isolated particles with DNase, RNase inhibitor, or both DNase and RNase inhibitors. In certain embodiments, the step of performing one or more steps to mitigate adverse factors is achieved by a step of treating the biological sample with RNase inhibitor before isolating the particles.
  • the present invention is a nucleic acid sample obtained from a biological sample by the any of above described methods.
  • the nucleic acid sample thus obtained can be used in various applications.
  • the above method and resulting nucleic acid sample are used for aiding in the diagnosis of a subject by determining the presence or absence of a biomarker within the nucleic acid sample that is associated with a known disease or other medical condition.
  • the above method and resulting nucleic acid sample are used for monitoring the progress or reoccurrence of a disease or other medical condition in a subject by determining the presence or absence of a biomarker with in the sample that is associated with the progress or reoccurrence of a known stage or the reoccurrence of a disease or other medical condition.
  • the above method and resulting nucleic acid sample are used in the evaluation of treatment efficacy for a subject undergoing or contemplating treatment for a disease or other medical condition by determining the presence or absence of a biomarker within the sample that is associated with treatment efficacy for the subject undergoing or contemplating treatment for a disease or other medical condition.
  • the biomarker detected in the above applications is a nucleic acid corresponding to any one or more of the genes consisting of EGFR, BRAF, KLK3, 18S, GAPDH, HPRT1, GUSB, ACTB, B2M, RPLP0, HMBS, TBP, PGK1, UBC, PPIA, ALCAM, C5AR1, CD160, CD163, CD19, CD1A, CD1C, CD1D, CD2, CD209, CD22, CD24, CD244, CD247, CD28, CD37, CD38, CD3D, CD3G, CD4, CD40, CD40LG, CD5, CD6, CD63, CD69, CD7, CD70, CD72, CD74, CD79A, CD79B, CD80, CD83, CD86, CD8A, CD8B, CD96, CHST10, COL1A1, COL1A2, CR2, CSF1R, CTLA4, DPP4, ENG, FAS, FCER1A, FCER2,
  • kits for use in the above methods may include RNase inhibitor in a quantity sufficient to mitigate adverse factors that prevent or might prevent high quality nucleic acid extraction, and an RNA purification reagent.
  • the kit may optionally further include a lysis buffer, DNase, or instructions for using the kit and reagent in it in the extraction of nucleic acids from isolated particles.
  • FIGS. 1A , 1 B and 1 C are Bioanlayzer plots depicting the analysis of nucleic acids extracted from particles isolated from serum samples as described in Examples 1, 2 and 3, respectively.
  • the pseudogel in FIG. 1A depicts the content of the same nucleic acid extraction as depicted in the Bioanalyzer plot of FIG. 1A .
  • the plots and the pseudogel were generated by an RNA pico chip run on an Agilent Bioanalyzer.
  • FIG. 2 is a Bioanalyzer plot depicting the analysis of nucleic acids extracted from particles isolated from a urine sample, as described in Example 5 below, and a pseudogel depicting the content of the same nucleic acid extraction. The plot and the pseudogel were generated by an Agilent Bioanalyzer.
  • FIG. 3 is a Bioanalzyer plot depicting the analysis of nucleic acids extracted from particles isolated from serum samples in group A, Example 4 (20,000 g centrifugation speed).
  • FIG. 4 is a Bioanalzyer plot depicting the analysis of nucleic acids extracted from particles isolated from serum samples in group B, Example 5 (120,000 g centrifugation speed).
  • FIG. 5 is a plot depicting the comparison of Ct values for genes analyzed with the Taqman PCR array as between group A (Y-axis) and group B (X-axis) in Example 4.
  • cell-derived vesicles are heterogeneous in size with diameters ranging from about 10 nm to about 5000 nm.
  • “exosomes” have diameters of approximately 30 to 100 nm, with shedding microvesicles and apoptotic bodies often described as larger (Orozco and Lewis, 2010).
  • Exosomes, shedding microvesicles, microparticles, nanovesicles, apoptotic bodies, nanoparticles and membrane vesicles co-isolate using various techniques and will, therefore, collectively be referred to throughout this specification as “microvesicles” unless otherwise expressly denoted.
  • nucleic acid-containing particles e.g., RNA-protein complexes and DNA-protein complexes
  • RNA-protein complexes and DNA-protein complexes may co-isolate with microvesicles using the various methods and techniques described herein.
  • the generic term “particles” will be used herein to refer to microvescles, RNA-protein complexes, DNA-protein complexes, and any other nucleic acid-containing particles that could be isolated according to the methods and techniques described herein.
  • the methods and techniques described herein are equally applicable to the isolation of RNA-protein complexes, DNA-protein complexes, or other nucleic acid-containing particles, and microvesicles of all sizes (either as a whole, as select subsets, or as individual species).
  • the present invention is partly based on the discovery that lower centrifugation speeds can achieve similar results as higher centrifugation speeds during nucleic acid-containing particle isolation.
  • the present invention is directed to novel methods for isolating particles from a biological sample and extracting nucleic acids from the isolated particles.
  • the nucleic acid extractions obtained by the methods described herein may be useful for various applications in which high quality nucleic acid extractions are required or preferred.
  • high quality nucleic acid extraction means an extraction in which one is able to detect 18S and 28S rRNA, preferably in a ratio of approximately 1:1 to approximately 1:2; and more preferably, approximately 1:2.
  • high quality nucleic acid extractions obtained by the methods described herein will also have an RNA integrity number of greater than or equal to 5 for a low protein biological sample (e.g., urine), or greater than or equal to 3 for a high protein biological sample (e.g., serum), and a nucleic acid yield of greater than or equal to 50 pg/ml from a 20 ml low protein biological sample or a 1 ml high protein biological sample.
  • RNA degradation can adversely affect downstream assessment of the extracted RNA, such as in gene expression and mRNA analysis, as well as in analysis of non-coding RNA such as small RNA and microRNA.
  • the new methods described herein enable one to extract high quality nucleic acids from particles isolated from a biological sample so that an accurate analysis of nucleic acids within the particles can be carried out.
  • the novel methods include, for example, the steps of obtaining a biological sample; isolating nucleic acid-containing particles from the biological sample by one or more centrifugation steps; mitigating or removing adverse factors that prevent high quality extraction of nucleic acids from the sample; and extracting nucleic acids from the isolated particles; followed, optionally, by nucleic acid analysis.
  • the centrifugation step or steps may be performed at relatively low speeds as compared to traditional methods of isolating particles from biological samples by centrifugation. None of the centrifugation steps in the inventive methods described herein may exceed about 200,000 g.
  • Suitable centrifugation speeds are up to about 200,000 g; for example from about 2,000 g to less than about 200,000 g. Speeds of above about 15,000 g and less than about 200,000 g or above about 15,000 g and less than about 100,000 g or above about 15,000 g and less than about 50,000 g are preferred. Speeds of from about 18,000 g to about 40,000 g or about 30,000 g; and from about 18,000 g to about 25,000 g are more preferred. Particularly preferred is a centrifugation speed of about 20,000 g.
  • the methods described herein may be used with a variety of commercially available centrifuge machines and for the purpose of isolating various species of particles.
  • a person of skill in the art will be able to use the well known K-factor to optimize the centrifugation parameters for a particular centrifuge device selected for use in the method.
  • the K-factor which denotes the clearing factor of a centrifuge rotor at maximum rotation speed, may be used to determine the time (“T”) required for pelleting a fraction with a known sedimentation coefficient (“S”).
  • T time
  • S sedimentation coefficient
  • the K-factor can be calculated by the following formula:
  • r max is the maximum radius from the centrifuge's axis of rotation
  • r min is the minimum radius from the axis of rotation.
  • the r max and r min are usually available from the centrifuge manufacturer.
  • RPM is the speed in revolutions per minute.
  • the K-factor is related to the sedimentation coefficient S by the formula:
  • T is the time to pellet a certain particle in hours.
  • S is a known constant for a certain particle, this relationship can be used to interconvert between different rotors using the following formula:
  • T 1 is the time to pellet in one rotor
  • K 1 is the K-factor of that rotor
  • K 2 is the K-factor of the other rotor
  • T 2 the time to pellet in the other rotor. If one knows K 1 , T 1 , and can calculate K 2 , then T 2 may be determined. In this manner, one does not need access to the exact centrifuge rotor cited in a particular protocol, as long as the K-factor can be calculated. If the sedimentation constant (S) is unknown for a particular substance to be pelleted, then one of skill in the art may determine T 2 based on empirical data as to T 1 for the same substance and calculation of K 2 for the different rotor.
  • S sedimentation constant
  • suitable K factors are within the range of about 300 to about 1000; preferably within the range of about 400 to about 600; and more preferably about 520.
  • suitable times for centrifugation are from about 5 minutes to about 2 hours, for example, from about 10 minutes to about 1.5 hours, or more preferably from about 15 minutes to about 1 hour. A time of about 0.5 hours is sometimes preferred.
  • the biological sample it is sometimes preferred to subject the biological sample to centrifugation at about 20,000 g for about 0.5 hours.
  • the above speeds and times can suitably be used in any combination (e.g., from about 18,000 g to about 25,000 g, or from about 30,000 g to about 40,000 g for about 10 minutes to about 1.5 hours, or for about 15 minutes to about 1 hour, or for about 0.5 hours, and so on).
  • the centrifugation step or steps may be carried out at below-ambient temperatures, for example at about 0-10° C., preferably about 1-5° C., e.g., about 3° C. or about 4° C.
  • the term “biological sample” refers to a sample that contains biological materials such as a DNA, a RNA and a protein.
  • the biological sample may suitably comprise a bodily fluid from a subject.
  • the bodily fluids can be fluids isolated from anywhere in the body of the subject, preferably a peripheral location, including but not limited to, for example, blood, plasma, serum, urine, sputum, spinal fluid, cerebrospinal fluid, pleural fluid, nipple aspirates, lymph fluid, fluid of the respiratory, intestinal, and genitourinary tracts, tear fluid, saliva, breast milk, fluid from the lymphatic system, semen, cerebrospinal fluid, intra-organ system fluid, ascitic fluid, tumor cyst fluid, amniotic fluid and combinations thereof.
  • the preferred body fluid for use as the biological sample is urine.
  • the preferred body fluid is serum.
  • the preferred body fluid is cerebrospinal fluid.
  • a sample volume of about 0.1 ml to about 30 ml fluid may be used.
  • the volume of fluid may depend on a few factors, e.g., the type of fluid used.
  • the volume of serum samples may be about 0.1 ml to about 2 ml, preferably about 1 ml.
  • the volume of urine samples may be about 10 ml to about 30 ml, preferably about 20 m1.
  • the term “subject” is intended to include all animals shown to or expected to have nucleic acid-containing particles.
  • the subject is a mammal, a human or nonhuman primate, a dog, a cat, a horse, a cow, other farm animals, or a rodent (e.g. mice, rats, guinea pig. etc.).
  • a human subject may be a normal human being without observable abnormalities, e.g., a disease.
  • a human subject may be a human being with observable abnormalities, e.g., a disease. The observable abnormalities may be observed by the human being himself, or by a medical professional.
  • the term “subject”, “patient”, and “individual” are used interchangeably herein.
  • the biological sample may be pre-processed before isolating nucleic acid-containing particles.
  • a pre-processing step is preferred.
  • a urine sample may be pre-processed to obtain urinary nucleic acid-containing particles.
  • the pre-processing may be achieved by techniques known in the art such as differential centrifugation or filtration.
  • urine samples may undergo a first centrifugation step of about 300 g to get rid of large particles and debris in the samples.
  • Urine samples may then undergo a second centrifugation step of about 5,000 g to about 20,000 g (larger volume centrifuged-higher k-factor) to get rid of unwanted particles that did not pellet in the previous centrifugation step, but without pelleting nucleic acid-containing particles that are desired in the final analysis.
  • urine samples may further undergo a filtration step, e.g., 0.8 ⁇ m, 0.45 ⁇ m, or 0.22 ⁇ m filtration step to further rid the sample of unwanted materials.
  • urine samples may be pre-processed by a filtration step without first undergoing the one or more of the centrifugation steps.
  • the biological sample may be pre-processed by centrifuging at a low speed of about 100-500 g, preferably about 250-300 g, to remove large unwanted particles and debris in the sample.
  • the biological sample may be pre-processed by centrifuging at a higher speed of about 10,000-20,000 g, preferably 15,000-19,000 g, to remove unwanted particles and substances in the sample.
  • the biological sample may be centrifuged first at the lower speed and then at the higher speed. If desired, further suitable centrifugation pre-processing steps may be carried out. For example, the step of centrifugation may be repeated for further pre-processing the samples.
  • the biological sample may be filtered.
  • a filter having a size in the range about 0.1 to about 1.0 ⁇ m may be employed, preferably about 0.5 to about 1.0 ⁇ m, e.g. about 0.7 ⁇ m or about 0.8 ⁇ m.
  • the isolation step is advantageous for the extraction of high quality nucleic acids from a biological sample for the following reasons: 1) extracting nucleic acids from particles provides the opportunity to selectively analyze disease- or tumor-specific nucleic acids, which may be obtained by isolating disease- or tumor-specific particles apart from other particles within the fluid sample; 2) nucleic acid-containing particles such as microvesicles produce significantly higher yields of nucleic acid species with higher integrity as compared to the yield/integrity obtained by extracting nucleic acids directly from the fluid sample without first isolating microvesicles; 3) scalability, e.g.
  • the sensitivity can be increased by pelleting more nucleic acid-containing particles from a larger volume of serum; 4) purer nucleic acids in that protein and lipids, debris from dead cells, and other potential contaminants and PCR inhibitors are excluded from the pellets before the nucleic acid extraction step; and 5) more choices in nucleic acid extraction methods as pellets are of much smaller volume than that of the starting serum, making it possible to extract nucleic acids from these pellets using small volume column filters.
  • the method of isolating particles from a body fluid and extracting nucleic acids from the isolated particles may comprise the steps of: removing cells from the body fluid either by low speed centrifugation and/or filtration though a 0.8 ⁇ m filter; centrifuging the supernatant/filtrate at about 20,000 g for about 0.5 hour at about 4° C. using about 1 ml sample volume; treating the pellet with a pre-lysis solution, e.g., an RNase inhibitor and/or a pH buffered solution and/or a protease enzyme in sufficient quantities (as described below); and lysing the pellet for nucleic acid extraction.
  • the process of isolating particles and extracting high quality nucleic acids may be achieved within 90 minutes.
  • nucleic acid may be extracted from the pelleted particles.
  • the particles may first be lysed.
  • the lysis of particles such as microvesicles in the pellet and extraction of nucleic acids may be achieved with various methods known in the art.
  • the lysis and extraction steps may be achieved using a commercially available Qiagen RNeasy Plus kit.
  • the lysis and extraction steps may be achieved using a commercially available Qiagen miRNeasy kit.
  • the nucleic acid extraction may be achieved using phenol:chloroform according to standard procedures and techniques known in the art.
  • the novel nucleic acid extraction methods include the step of removing or mitigating adverse factors that prevent high quality nucleic acid extraction from a biological sample.
  • adverse factors are heterogeneous in that different biological samples may contain various species of adverse factors.
  • factors such as excessive DNA may affect the quality of nucleic acid extractions from such samples.
  • factors such as excessive endogenous RNase may affect the quality of nucleic acid extractions from such samples.
  • Many agents and methods may be used to remove these adverse factors. These methods and agents are referred to collectively herein as an “extraction enhancement operations.”
  • the extraction enhancement operation may involve the addition of nucleic acid extraction enhancement agents to the biological sample.
  • extraction enhancement agents as defined herein may include, but are not limited to, an RNase inhibitor such as Superase-In (commercially available from Ambion Inc.) or RNaseINplus (commercially available from Promega Corp.), or other agents that function in a similar fashion; a protease (which may function as an RNase inhibitor); DNase; a reducing agent; a decoy substrate such as a synthetic RNA and/or carrier RNA; a soluble receptor that can bind RNase; a small interfering RNA (siRNA); an RNA binding molecule, such as an anti-RNA antibody, a basic protein or a chaperone protein; an RNase denaturing substance, such as a high osmolarity solution, a detergent, or a combination thereof.
  • an RNase inhibitor such as Superase-In (commercially available from Ambion Inc.) or RNaseINplus (commercially available from Promega Corp
  • enhancement agents may exert their functions in various ways, e.g., through inhibiting RNase activity (e.g., RNase inhibitors), through a ubiquitous degradation of proteins (e.g., proteases), or through a chaperone protein (e.g., a RNA-binding protein) that binds and protects RNAs.
  • RNase activity e.g., RNase inhibitors
  • a ubiquitous degradation of proteins e.g., proteases
  • a chaperone protein e.g., a RNA-binding protein
  • rRNA ribosomal RNA
  • the extracted nucleic acid comprises RNA.
  • the RNA is preferably reverse-transcribed into complementary DNA (cDNA) before further amplification.
  • cDNA complementary DNA
  • Such reverse transcription may be performed alone or in combination with an amplification step.
  • a method combining reverse transcription and amplification steps is reverse transcription polymerase chain reaction (RT-PCR), which may be further modified to be quantitative, e.g., quantitative RT-PCR as described in U.S. Pat. No. 5,639,606, which is incorporated herein by reference for this teaching.
  • Another example of the method comprises two separate steps: a first of reverse transcription to convert RNA into cDNA and a second step of quantifying the amount of cDNA using quantitative PCR.
  • RNAs extracted from nucleic acid-containing particles using the methods disclosed herein include many species of transcripts including, but not limited to, the transcripts that correspond to those for GAPDH, BRAF, KLK3, EGFR, and ribosomal 18S rRNA.
  • Nucleic acid amplification methods include, without limitation, polymerase chain reaction (PCR) (U.S. Pat. No. 5,219,727) and its variants such as in situ polymerase chain reaction (U.S. Pat. No. 5,538,871), quantitative polymerase chain reaction (U.S. Pat. No. 5,219,727), nested polymerase chain reaction (U.S. Pat. No.
  • PCR polymerase chain reaction
  • U.S. Pat. No. 5,219,727 in situ polymerase chain reaction
  • quantitative polymerase chain reaction U.S. Pat. No. 5,219,727
  • nested polymerase chain reaction U.S. Pat. No.
  • the analysis of nucleic acids present in the isolated particles is quantitative and/or qualitative.
  • the amounts (expression levels), either relative or absolute, of specific nucleic acids of interest within the isolated particles are measured with methods known in the art (described below).
  • the species of specific nucleic acids of interest within the isolated particles, whether wild type or variants, are identified with methods known in the art.
  • the present invention also includes various uses of the new methods of nucleic acid extraction from a biological sample for (i) aiding in the diagnosis of a subject, (ii) monitoring the progress or reoccurrence of a disease or other medical condition in a subject, or (iii) aiding in the evaluation of treatment efficacy for a subject undergoing or contemplating treatment for a disease or other medical condition; wherein the presence or absence of one or more biomarkers in the nucleic acid extraction obtained from the method is determined, and the one or more biomarkers are associated with the diagnosis, progress or reoccurrence, or treatment efficacy, respectively, of a disease or other medical condition.
  • the one or more biomarkers can be one or a collection of genetic aberrations, which is used herein to refer to the nucleic acid amounts as well as nucleic acid variants within the nucleic acid-containing particles.
  • genetic aberrations include, without limitation, over-expression of a gene (e.g., an oncogene) or a panel of genes, under-expression of a gene (e.g., a tumor suppressor gene such as p53 or RB) or a panel of genes, alternative production of splice variants of a gene or a panel of genes, gene copy number variants (CNV) (e.g., DNA double minutes) (Hahn, 1993), nucleic acid modifications (e.g., methylation, acetylation and phosphorylations), single nucleotide polymorphisms (SNPs), chromosomal rearrangements (e.g., inversions, deletions and duplications), and mutations (insertions, deletions, duplications, miss
  • nucleic acid modifications can be assayed by methods described in, e.g., U.S. Pat. No.
  • methylation profiles may be determined by Illumina DNA Methylation OMA003 Cancer Panel.
  • SNPs and mutations can be detected by hybridization with allele-specific probes, enzymatic mutation detection, chemical cleavage of mismatched heteroduplex (Cotton et al., 1988), ribonuclease cleavage of mismatched bases (Myers et al., 1985), mass spectrometry (U.S. Pat. Nos.
  • nucleic acid sequencing single strand conformation polymorphism (SSCP) (Orita et al., 1989), denaturing gradient gel electrophoresis (DGGE)(Fischer and Lerman, 1979a; Fischer and Lerman, 1979b), temperature gradient gel electrophoresis (TGGE) (Fischer and Lerman, 1979a; Fischer and Lerman, 1979b), restriction fragment length polymorphisms (RFLP) (Kan and Dozy, 1978a; Kan and Dozy, 1978b), oligonucleotide ligation assay (OLA), allele-specific PCR (ASPCR) (U.S. Pat. No.
  • DGGE denaturing gradient gel electrophoresis
  • TGGE temperature gradient gel electrophoresis
  • RFLP restriction fragment length polymorphisms
  • OLA oligonucleotide ligation assay
  • ASPCR allele-specific PCR
  • gene expression levels may be determined by the serial analysis of gene expression (SAGE) technique (Velculescu et al., 1995).
  • SAGE serial analysis of gene expression
  • biomarkers may be associated with the presence or absence of a disease or other medical condition in a subject. Therefore, detection of the presence or absence of such biomarkers in a nucleic acid extraction from isolated particles, according to the methods disclosed herein, may aid diagnosis of the disease or other medical condition in the subject. For example, as described in WO 2009/100029, detection of the presence or absence of the EGFRvIII mutation in nucleic acids extracted from microvesicles isolated from a patient serum sample may aid in the diagnosis and/or monitoring of glioblastoma in the patient. This is so because the expression of the EGFRvIII mutation is specific to some tumors and defines a clinically distinct subtype of glioma (Pelloski et al., 2007).
  • detection of the presence or absence of the TMPRSS2-ERG fusion gene and/or PCA-3 in nucleic acids extracted from microvesicles isolated from a patient urine sample may aid in the diagnosis of prostate cancer in the patient.
  • biomarkers may help disease or medical status monitoring in a subject. Therefore, the detection of the presence or absence of such biomarkers in a nucleic acid extraction from isolated particles, according to the methods disclosed herein, may aid in monitoring the progress or reoccurrence of a disease or other medical condition in a subject.
  • MMP matrix metalloproteinase
  • the determination of matrix metalloproteinase (MMP) levels in nucleic acids extracted from microvesicles isolated from an organ transplantation patient may help to monitor the post-transplantation condition, as a significant increase in the expression level of MMP-2 after kidney transplantation may indicate the onset and/or deterioration of post-transplantation complications.
  • MMP-9 after lung transplantation suggests the onset and/or deterioration of bronchiolitis obliterans syndrome.
  • biomarkers have also been found to influence the effectiveness of treatment in a particular patient. Therefore, the detection of the presence or absence of such biomarkers in a nucleic acid extraction from isolated particles, according to the methods disclosed herein, may aid in evaluating the efficacy of a given treatment in a given patient.
  • biomarkers e.g., mutations in a variety of genes, affect the effectiveness of specific medicines used in chemotherapy for treating brain tumors.
  • the identification of these biomarkers in nucleic acids extracted from isolated particles from a biological sample from a patient may guide the selection of treatment for the patient.
  • kits for use in the new methods disclosed herein are comprised of the following components: RNase inhibitor in quantity sufficient to mitigate adverse factors that prevent or might prevent high quality nucleic acid extraction; RNA purification reagent; optionally, lysis buffer; optionally, DNase; and optionally, instructions for using the foregoing reagents in the extraction of nucleic acids from isolated particles.
  • RNase inhibitor in quantity sufficient to mitigate adverse factors that prevent or might prevent high quality nucleic acid extraction
  • RNA purification reagent in quantity sufficient to mitigate adverse factors that prevent or might prevent high quality nucleic acid extraction
  • RNA purification reagent optionally, lysis buffer
  • DNase optionally, instructions for using the foregoing reagents in the extraction of nucleic acids from isolated particles.
  • the RNA purification reagent helps to purify the released nucleic acids.
  • the lysis buffer helps to break open microvesicles so that their nucleic acid contents are released.
  • DNase may help enhance the quality of the extracted nucleic acids.
  • kits may also comprise instructions that detail the steps as appropriate for using the kit components in connection with the extraction of nucleic acids from isolated particles.
  • the pellet was treated with 8 ⁇ l SuperaseIn (20 units/ ⁇ l) for 1 minute and then re-suspended in RLT buffer plus 10 ⁇ l/ml betamercaptoethanol and processed using the Qiagen RNeasy Plus kit which features a DNA removal column.
  • the nucleic acids were eluted in 16 ⁇ l nuclease-free H 2 O.
  • RNA Pico Chip on an Agilent Bioanalyzer. As shown in FIG. 1A , we detected the presence of the 18S and 28S rRNA in the extraction. The RNA Integrity Number (RIN), as calculated by the Bioanalyzer's software, was 8.5. In addition, in the extracted nucleic acids, we detected the presence of RNAs corresponding to the GAPDH, BRAF, and 18S RNA genes. We used 12 ⁇ l of the extracted RNA and reverse transcribed the RNA into cDNA using a Sensiscript kit (Qiagen). We then used 2 ⁇ l of the resulting cDNA product as templates to perform Real-time PCR.
  • RIN RNA Integrity Number
  • the primers used for the RT-PCR are commercially available from Applied Biosystems, as follows: Human GAPDH (part number 4326317E); BRAF (part number Hs00269944_m1); 18S rRNA (part number Hs99999901_s1). Each sample was run in triplicate on the PCR plate. The Cl values from the RT-PCR investigation are presented as average ⁇ SD. The Ct values for GAPDH, BRAF and 18S rRNA are 30.84 ⁇ 0.08, 36.76 ⁇ 0.22, and 15.09 ⁇ 0.21, respectively.
  • the nucleic acids extracted from the pelleted particles contained 18S and 28S rRNA.
  • the quality of the nucleic acids produced a RIN of 8.5.
  • the extracted nucleic acids contain RNAs corresponding to at least GAPDH, BRAF and 18S rRNA genes, suggesting that the extracted nucleic acids from serum particles may include RNAs corresponding to many other genes.
  • Example 2 We obtained a 1 ml frozen serum sample from the same normal, healthy human volunteer as in Example 1 and filtered the serum through a 0.8 ⁇ m filter (Millipore) and the filtrate was then stored at ⁇ 80° C. for 24 hours.
  • the frozen sample was thawed on ice, and transferred into a 1.5 ml Eppendorf tube containing 8 ⁇ l SuperaseIn (Ambion Inc.). After the 20,000 g, 0.5 hour centrifugation step, the supernatant was set aside for further extraction as detailed in Example 3 below.
  • the pellet was used for nucleic acid extraction employing a modified miRNeasy RNA extraction protocol. This modified protocol was more efficient at capturing the small RNAs (e.g., less than 200 nucleotides) than the manufacturer's protocol contained in the RN easy Plus kit used in Example 1.
  • the pellet was mixed with 50 ⁇ L of the DNase/SuperaseIn mixture as mentioned above and incubated at room temperature for 20 min in the centrifuge tube. Then 700 ⁇ l Qiazol lysis buffer (Qiagen) was added to each sample in the centrifuge tube and mixed by pipetting up and down 15 times to dissolve/re-suspend the pellet. The suspended pellet mixture was immediately transferred to an Eppendorf tube. Further nucleic acid extraction was then performed in a PCR hood. The tube with the pellet mixture was vortexed briefly and incubated at room temperature for 2-4 minutes before 140 ⁇ l chloroform was added into the tube containing the mixture.
  • Qiazol lysis buffer Qiagen
  • the tube was then capped, shaken vigorously for 20 seconds, incubated at room temperature for 2-3 minutes, and centrifuged for 15 minutes at 12,000 g at 4° C.
  • the upper aqueous phase was transferred to a new collection tube into which, 1.5 volumes (usually 600 ⁇ l) of 100% ethanol was added and mixed thoroughly by pipetting up and down several times.
  • the nucleic acids on the column were then washed three times as follows: 1) 700 ⁇ L Buffer RWT was added onto the RNeasy MinElute spin column and centrifuged for 15 seconds at 8500 g to wash the column with the flow-through discarded; 2) 500 ⁇ L Buffer RPE was added onto the RNeasy MinElute spin column and centrifuged for 15 seconds at 8500 g to wash the column with the flow-through discarded; 3) repeat the Buffer RPE wash step except that the column was centrifuged for 2 minutes at 8500 g to dry the RNeasy Mini spin column membrane.
  • the RNeasyMinElute spin column was inserted into a new 2 ml collection tube and centrifuged at 14000 g for 5 minutes to further dry the column membrane.
  • the dried column was inserted into another new 1.5 ml collection tube and 16 ⁇ L RNase-free water was added onto the dried column membrane and incubated for 1 minute at room temperature.
  • the nucleic acids were eluted by centrifugation for 1 minute at 8500 g.
  • the volume of the eluted nucleic acids was about 14 ⁇ l.
  • the reverse transcription was performed in a verity PCR machine under the following conditions: 25° C. for 10 minutes, 42° C. for 70 minutes, 85° C. for 5 minutes, and was held in 4° C. before the reaction was stored at ⁇ 20° C.
  • the primers used for the RT-PCR are commercially available from Applied Biosystems, as follows: Human GAPDH (part number 4326317E); BRAF (part number Hs00269944_m1); 18S rRNA (part number Hs99999901_s1); EGFR (part number HS01076088_m1). We repeated the real time-PCR experiments two times for each gene. The Ct values are shown in Table 2.
  • nucleic acid-containing particles from a serum sample.
  • the nucleic acids extracted from the isolated particles contained 18S and 28S rRNA, as well as small RNAs. Further, the extracted nucleic acids contained RNAs for at least GAPDH, BRAF and 18S rRNA genes, suggesting that the extracted nucleic acids from serum particles may include RNAs corresponding to many other genes.
  • Example 2 we started with the supernatant obtained in Example 2 after centrifuging the 1 ml serum sample at 20,000 g for 0.5 hour. The supernatant was further ultracentrifuged at 120,000 g for 80 minutes at 4-8° C. (Optima Max-XP Benchtop ultracentrifuge from Beckman). The deceleration was set at 7. Nucleic acids were then extracted from the pellet following the same protocol as detailed above in Example 2 starting from a treatment with DNase and SuperaseIn mixture. We analyzed the profile of the extracted nucleic acids, and performed reverse transcription and real time PCR analysis of the same four genes as in Example 2.
  • Example 2 we also detected the RNAs corresponding to the four genes GAPDH, BRAF, 18S rRNA, and EGFR.
  • the Ct values are shown in Table 3.
  • nucleic acid-containing particles from the supernatant obtained in Example 2 after serum centrifugation at 20,000 g for 0.5 hour.
  • the nucleic acids extracted from the particles pelleted from the supernatant contained more abundant small RNAs than the nucleic acids extracted from the particles initially pelleted in Example 2. Further, the extracted nucleic acids contained RNAs for at least GAPDH, BRAF and 18S rRNA genes, suggesting that the extracted nucleic acids from supernatant may include RNAs corresponding to many other genes.
  • group A the serum samples were centrifuged at 20,000 g for 0.5 hour and the pellet was used for nucleic acid extraction employing a modified miRNeasy RNA extraction protocol as described in Example 2.
  • group B the serum samples were centrifuged at 120,000 g for 80 minutes and the pellet was used for nucleic acid extraction employing a modified miRNeasy RNA extraction protocol as described in Example 2.
  • RNA extracted from particles pelleted from each of the serum samples was each reversed transcribed into cDNA using the VILOTM kit from Invitrogen as described in Example 2, and then analyzed using the TagMan® array 96 Human Cell Surface Markers PCR plate from Applied Biosystems according to the manufacturer's protocol.
  • nucleic acid-containing particles from the serum sample by centrifugation at either 20,000 g for 0.5 hour or 120,000 g for 80 minutes.
  • the nucleic acids extracted from the isolated particles contained both 18S and 28S rRNA.
  • the mRNA content obtained with a 20,000 g centrifugation speed was similar to the mRNA content obtained with a 120,000 g centrifugation speed.
  • the extracted nucleic acids from each of the pellets contained mRNAs corresponding to most of the genes tested using the Taqman array.
  • the primers used for the RT-PCR are commercially available from Applied Biosystems, as follows: Human GAPDH (part number 4326317E); KLK3 (part number Hs03063374_m1); 18S rRNA (part number Hs99999901_s1). Each sample was run in triplicate on the PCR plate. The Ct values from the RT-PCR investigation are presented as average ⁇ SD. The Ct values for GAPDH, KLK3 and 18S rRNA are 26.96 ⁇ 0.02, 30.18 ⁇ 0.01, and 12.22 ⁇ 0.15, respectively.
  • nucleic acid-containing particles from urine samples.
  • the nucleic acids extracted from the pelleted particles contained 18S and 28S rRNA.
  • the quality of the nucleic acids produced a RIN of 9.1.
  • the extracted nucleic acids contain RNAs for at least GAPDH, KLK3 and 18S rRNA genes, suggesting that the extracted nucleic acids from urine particles may include RNAs corresponding to many other genes.

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